Help on BAC DNA prep!

In article <19970929221400.SAA20314 at ladder01.news.aol.com>, wzhao68 at aol.com says...
>>Hello there, I am having problem with BAC DNA preparation. I've tried
>miniprep protocol from Research Genetics and Qiagen's Midiprep kit. The
>miniprep never worked and Qiagen kit worked sometimes, but most of the time
>gave me genomic DNA.
>Could someone who has experience on this help me? Please e-mail me.
>Thanks in advance!
>David
>Children's Hospital
>
David,
We've done thousands of BAC minipreps and a "standard" alkaline lysis with a few
modifications works best for us (soybean DNA inserts). Use 2mL of a 5 mL overnight
culture, 100 ul of Maniatis etc soln I to resuspend cell pellet, 200 uL Maniatis
etc soln II freshly mixed just before use to lyse (lyse by inverting 6-8 times and
leave on ice no longer than 5 min). Neutralize with 150 uL 7.5M NH4OAc (soln not
older than 4 weeks), mix as with soln II, stand on ice 5 min, invert just before
centrifuging (room temp ok) not more than 8000 rpm (microcentrifuge) for 6-7 min.
If you just want to do digests to size inserts etc, respin super to clarify and then
precipitate nucleic acids with ethanol and resuspend in TE plus RNAase. If you want
to do sequecing with the DNA, treat the super (from spinning after neutralizing)
with RNAase, then extract with phenol cholorform 1x and chloroform 1x (1-200uL each
depending on how cloudy the super was) and precipitate the nucleic acids with
ethanol. I like to let the preps sit overnight at room temp and then spin 10-12 min
at room temp to pellet DNA. Can be scaled up for 50 mL preps. If you need really
clean DNA for a minilibrary or whatever then a CsCl gradient is necessary. Because
the BAC vector is single copy there is always a significant carryover of bacterial
DNA in a miniprep.
Laura Marek
lmarek at iastate.edu
Agronomy Department
Iowa State University